The Viability of Leptospires in the Summer Paddy Water1

Transcription

1 Japan. J. Microbiol. Vol. 10 (1), 51-57, 1966 The Viability of Leptospires in the Summer Paddy Water1 Eihyo Ryu and Chaw-King LIU Department of Veterinary Medicine, College of Agriculture, National Taiwan University, Taipei, Taiwan, China (Received for publication, February 10, 1966) ABSTRACT In connection with the possible role played by water buffaloes in transmitting leptospiral infection, surveys of ph and temperature of paddy water in Taiwan as well as investigation of their effects upon the viability of various leptospires were performed during the summer time. The ph values of paddy water at 122 places in 37 villages distributed in the southern, central and northern parts of Taiwan were surveyed. The ph values of the water of 8 (6.6%) places ranged from 6.4 to 6.8, that of 29 (23.8%) places varied between 7.0 and 7.5, and that of 85 (69.7%) places ranged from 7.6 to 7.8. The temperature of these paddy waters was surveyed for one week between July and August. Temperatures ranging from 40C to 43C were obtained from 8 out of 11 places in the south, 9 out of 11 places in the central section and 5 out of 10 places in the north. L. australis A lost its viability in paddy water within one day in the experiment performed in the fields between July and August in the southern, central and northern areas, while 2 out of 21 strains of leptospires (L. semarang and L. australis A) maintained their viability for one week in the experiment performed between August and September in Taipei. In laboratory experiments, L. australis A was killed in paddy water at 42C within 3 hours compared to 6 hours in L. semarang. The viability of both strains of leptospires was markedly influenced at -15C, while they maintained their viability for 7 to 14 days at temperatures ranging from 0C to 30C. Opportunities for exposure to leptospiral infection in water buffaloes are more abundant than that of the other animals due to their direct contact with paddy water and their habit of the water bathing. If water buffaloes are susceptible to leptospires, the organisms undoubtedly would be excreted in paddy fields, ponds and sewerage. Consequently, water buffalo could play 1 This study was supported by Grant No. AI BMA of Public Health Service Research Grant Program in U. S. Department of Health, Education, and Welfare. an important role in transmitting leptospires to other species of domestic animals as well as to human beings, particularly to farm workers. Therefore, the susceptibility of water buffaloes to leptospires could constitute a very important public health problem. However, our present knowledge as to whether water buffaloes are susceptible to leptospires is obscure. A survey of leptospirosis among domestic animals in Taiwan has been carried out by Young et al. [9], but the report was lacking in data concerning water buffaloes. However Yeh [8] isolated 51

2 52 E. RYU AND C. K. LIU leptospires from the kidneys of 11 (10.47%) of 105 wild rats in Taiwan, three of which were L. australis A and the other eight, L. javan.ica. Leptospires require an optimal temperature of 25C to 30C [1] to maintain their viability and 28.5C to 29.5C [3] for growth. They are susceptible to heat and are destroyed at 45C for 30 minutes [2]. As the average temperature of the summer paddy water in Taiwan is above 40C, it is questionable that leptospires are viable in the paddy water during this season. This point is considered to be related to the distribution of leptospirosis of domestic animals in the tropics and subtropics. Consequently, before initiating the study on the susceptibility of water buffaloes to leptospires, a survey of the viability of leptospires in paddy water and soil is necessary. It is known that temperature and ph are important factors influencing the viability of leptospires in water [2], but fertilizer [7] and ultraviolet rays [4] could affect the viability of leptospires in paddy water as well. This paper describes the results of a survey of the influence of water temperature and ph on the viability of leptospires during the summer, July to September. MATERIALS AND METHODS This survey was started about two weeks after the autumn rice seedling had been transplanted. The rice-planting season generally occurs between the middle of June and July. However, it was two to three weeks late in the central and northern areas of Taiwan this year (1965), because the harvest of spring rice had been delayed because of the average low air temperature. Since the rice-planting season begins earlier in the south, and is followed by the central region and then the north, the survey of the temperature and ph value of paddy water was started in that order. The temperature of paddy water was determined between one and two o'clock in the afternoon. Simultaneously, the ph value of paddy water was measured with a B. T. B. colorimeter. Experiments concerning the influence of paddy water on the viability of leptospires was performed both in the fields and in the laboratory. Field experiments. The paddy water samples having different ph values were collected, filtered through paper and 100ml of each sample then poured into a bottle. These samples were autoclaved for 15 minutes at 15 pounds pressure, and inoculated with 0.1ml of one-week-old culture of leptospires inorthof's medium. The inoculated samples were then distributed in 2ml amounts into 2ml ampoules. After sealing the ampoules by flame they were placed in silk nets, and then dropped into rice paddies. At regular intervals, ampoules were brought from the paddies to the laboratory and 0.1ml from each ampoule then aseptically inoculated into each of two tubes containing 2ml of Korthof's medium and incubated at 28C. Laboratory experiments. Screw-capped tubes containing 1.8ml of paddy water were inoculated with 0.2ml of a 1:100 dilution of a culture of L. australis A or L. semarang. The tubes were then divided into two groups: In one group, the tubes were placed into water baths at temperatures ranging from 38C to 44C at intervals of 2C. After incubation at the respective temperatures for 3 hours and 6 hours, 0.1ml from each of the tubes was inoculated into each of two tubes of Korthof's medium containing 2ml of medium per tube. They were then incubated at 28C. In another group, the tubes were kept in store-rooms at respective temperatures of -15C, 0C, 5C, 10C, 15C, 25C and 30C. They were re-

3 THE VIABILITY OF LEPTOSPIRES IN THE SUMMER PADDY WATER 53

4 54 E. RYU AND C. K. LIU moved from the store-rooms at regular intervals. The method of inoculation and cultivation used was essentially the same as that described in the preceding group. The rabbit sera used were all screeningtested [6], 10% of which was added to the Korthof's basal medium. In this study 21 strains comprised of 11 serotypes, viz., L. icterohaemorrhagiae, L. canicola, L. pyrogenes, L. hebdomadis, L. australis A, L. pomona, L. autumnalis, L. grippotyphosa, L. javanica, L. batavian and L. semarang were used. Among them 3 strains of L. australis A and 3 strains of L. javanica were isolated from the kidneys of wild rats in Taipei [8]. These strains were maintained by subculturing in Korthof's medium containing 10% of screening-tested rabbit serum. The cultures of leptospires were incubated at 28C for one to two weeks, and were examined for growth at 100 ~ magnification under dark field illumination. The number of leptospires seen was recorded in the same manner as described in previous papers [5, 6]. RESULTS The ph values of paddy water of 122 places in 37 villages distributed in the southern, central and northern parts of Taiwan were surveyed. Among them, the ph values of the water of 8 (6.6%) places ranged from 6.4 to 6.8, that of 29 (23.8%) places varied between 7.0 and 7.5, and that of 85 (69.7%) places ranged from 7.6 to 7.8. That is to say, as shown in Table 1, the ph value of about two thirds of paddy water samples examined ranged from 7.6 to 7.8. The ph of paddy water at rice-planting and weeding is influenced by that of the soil which is slightly acidic. However, it is generally elevated to a slight alkalinity several days later owing to the separation of water from the soil and an irrigation of stream water. No relation was found between the ph and the distribution of fertilizer prior to the weeding. As shown in Table 1, temperatures of paddy water ranging from 40C to 43C were obtained from 8 out of 11 places in the south, 9 out of 11 places in the central and 5 out of 10 places in the north. Weather is a very important factor influencing the temperature of paddy water. It is low on a cloudy day or on rainy days before and after typhoons. Showers after 2 o'clock p.m. do not markedly influence the temperature of paddy water on the following day, but the temperature of paddy water will be elevated Table 2. Influence of paddy water on the viability of L. australis A -: no organism per dark field (100 ~),+5: the greatest number of organisms per dark field (100 ~),+4: more than 100,+3: , +2: 11-30, }: less than 1 organism per field.

5 THE VIABILITY OF LEPTOSPIRES IN THE SUMMER PADDY WATER 55 Table 3. Temperature of paddy water in field experiments temp.: temperature. Table 4. Influence of paddy water to the viability of leptospires Symbols are the same as Table 2.

6 56 E. RYU AND C. K. LIU Table 5. Influence of temperature (38C to 44C) on the viability of L. australis A and L. semarang in paddy water in the laboratory Symbols are the same as Table 2. Table 6. Influence of temperature (-15C to 30C) on the viability of L. semarang and L. australis A in paddy water in the laboratory Symbols are the same as Table 2. to above 40C after a series of days without shower. A fall of the temperature of paddy water is seen following the growth of rice after September. I. australis A lost its viability in paddy water within one day in the experiment performed in the fields in the southern, central and northern areas (Table 2). The daily temperatures during the experimental periods are shown in Table 3. Viability was maintained for 7 days in 2 out of 21 strains of leptospires in the subsequent experiment performed in the northern area in August. However, L. semarang, one of these two strains, lost its viability within one day in the supplementary test (Table 4). The viability of leptospires in paddy water at temperatures ranging from 38C to 44C was examined in the laboratory. Only two strains of leptospires, L. australis A and L. semarang, were used in this experiment. As shown in Table 5, L. australis A was killed at 42C within 3 hours compared to 6 hours in L. semarang. The result of experiments at temperatures below 30C is recorded in Table 6. Both strains of leptospires were markedly influenced at -15C, while they maintained their viability for 7 days at temperature ranging from 0C to 30C. Two weeks' viability was maintained in L. semarang between 0C and 25C and in L. australis A

7 THE VIABILITY OF LEPTOSPIRES IN THE SUMMER PADDY WATER 57 between 15C and 25C. DISCUSSION The hottest season in Taiwan is from June to August. The temperature of paddy water rises markedly during this period. However, it is hot throughout the year except in winter in the central and northern areas. The rise in water temperature is limited to a few hours before and after noon. The viability of leptospires in paddy water within this time remains questionable. Throughout the summer, water buffaloes are in contact with paddy water only during the period between the harvesting of the spring rice and planting of autumn rice seedlings, which generally takes place between June and July. As for the farmer, the contact-period is extended to the weeding. The survival of leptospires, if they were excreted in paddy water by buffalo-carriers in this hottest season when air temperatures are high and that of paddy water is generally above 40C during this period, has been experimentally proved difficult. Only 1 out of 15 strains of leptospires remained alive from the 23rd to the 30th of August in an experiment in the fields performed in Taipei. In a subsequent experiment, only 1 out of 7 strains of leptospires, isolated from the kidneys of wild rats in Taipei, maintained its viability from the 8th to the 15th of September. The difficulty in maintaining their viability for 7 days in paddy water during this period may be attributed to the gradual falling temperature. Most ph values of rice-planted paddy water in Taiwan are between 7 and 8. The number of weakly acidic samples are very few. Thus it is clear that ph is not related to the leptospirocidal action of paddy water. It is thought that temperature alone or in cooperation with other unknown factors may be responsible for this action. Although leptospires lose their viability in the paddy water above 40C, they are able to maintain their viability in the paddy water between 0C and 30C for one or two weeks. It may be probable that ultraviolet rays exert some effect upon the survival of leptospires in paddy water. REFERENCES [1] Chang, S. L Studies on Leptospira icterohaenzorrhagiae. III. The growth rate of, and some biochemical observations on Leplospira icterohaenzorrhagiae in culture. J. Infect. Dis. 81: [2] Chang, S. L., Buckingham, M., and Tailor, N. P Studies on Leptospira icterohaemorrhagiae. IV. Survival in water and sewerage: Destruction in water by halogen compounds, synthetic detergents, and heat. J. Infect. Dis. 82: [3] Ellinghausen, H. C Nephelometry and a nephelo-cttlture flask used in measuring growth of leptospires. 20: Amer. J. Vet. Res. [4] Polanen, Th. O. E Action in vitro et in vivo de divers facteurs physiques sur les leptospires. Rev. Belg. Sci. Med. 13: 71. [5] Ryu, E Inhibitory action of rabbit serum on the growth of Leptospira. Bull. Inst. Zool., Academica Sinica 3: 1-8. [ 6] Ryu, E Screening test of animal sera for the cultivation of leptospires. Can. J. Comp. Med. Vet. Sci. 29: [7] Tohyama, Y Result of the prophylaxis of Weil's disease (Spirochaetosis icterohaernorrhagica) experimented in Kagoshima Prefecture. Sci. Rep. Inst. Infect. Dis. Tokyo Univ. 6: [8] Ych, Y. C Isolation of leptospires from wild rats in Taiwan. Japan. J. Vet. Med. Assoc. 17: (in Japanese) [9] Young, S. S., Yeh, Y. C., Sheng, Y. S., Hsiao, C. T., and Wang, C. C Preliminary survey of leptospirosis in domestic animals on Taiwan. Mem. College Agr., Nat. Taiw. Univ. 7 (2):